Timothy Daniels, MLS(ASCP), MB, QLS, and Helmut Weigelin, MLS(ASCP) were instrumental in establishing this extraction method within our laboratory.

The present protocol follows the human care guidelines and with approval of the institutional review board (IRB) of the University of Michigan. A waiver of informed consent was obtained for this from IRB. There are no relevant inclusion or exclusion criteria for the patients involved.
1. Separation of cellular and cell-free components
NOTE: Three types of samples can be received for VRL diagnostic testing: vitreous (undilute; fluid from vitrectomy collected prior to beginning infusion), diluted vitreous within a cassette bag (Figure 1), and aqueous humor (fluid from the anterior chamber of the eye).
<ol>
	<li>For undiluted vitreous fluid, which is very viscous, dilute with 3-5 mL PBS to facilitate pipetting.</li>
	<li>For dilute vitreous within the cassette bag (Supplemental Figure 1), drain fluid into 50 mL conical tubes (one for every 45 mL of fluid).</li>
	<li>Aqueous humor is very low in volume (&#60;200 &#181;L). To ensure maximum recovery of cells, transfer the sample to a 1.5 mL tube, rinse the original 500 &#181;L microcentrifuge tube the sample was collected in with an equal volume of PBS, and add to the 1.5 mL tube.</li>
	<li>Centrifuge vitreous or aqueous fluid at 3,000 x g for 15 min at room temperature.</li>
	<li>Carefully remove the supernatant using a pipette, being careful not to disturb the pelleted cells.</li>
</ol>
2. DNA extraction from the cellular component
<ol>
	<li>Add an appropriate amount of cell lysis solution (300 &#181;L for small pellet, 1 mL or more for larger pellet) (see Table of Materials). Mix by pipetting up and down.</li>
	<li>When completely lysed, add 1/3 total volume of the protein precipitate solution (100 &#181;L for 300 &#181;L lysate) (see Table of Materials).</li>
	<li>Vortex vigorously for 20-30 s. Spin for 3 min at 3,000 x g at room temperature.</li>
	<li>Pipette 300 &#181;L of isopropanol into a clean microcentrifuge tube. Carefully pipette the supernatant from the previous step to the isopropanol tube.</li>
	<li>Add 1.5 &#181;L of 20 mg/mL glycogen (for 300 &#181;L lysis) (see Table of Materials).</li>
	<li>Mix well by inversion, then place the sample in a -20 &#176;C freezer for 1 h to overnight to aid in DNA precipitation.</li>
	<li>Centrifuge the 1.5 mL tube for 5 min at 3,000 x g at room temperature. Pipette off the isopropanol.</li>
	<li>Add 0.5 mL of 70% ethanol and invert the tube to wash the pellet.</li>
	<li>Spin for 1 min at 3,000 x g at room temperature and decant the ethanol. Repeat the ethanol wash once.</li>
	<li>Invert the tube on clean gauze to drain the solution and dry the pellet or decant. Quickly spin the DNA in a microcentrifuge and pipette the remaining drop of ethanol with a fine-tip disposable pipet tip (once no residual ethanol is left in the tube, the DNA should be dry and ready to hydrate in 5-10 min).</li>
	<li>Add 45 &#181;L of DNA hydration solution (see Table of Materials) to the dried DNA pellet.</li>
	<li>Place the tube in a heating block at 50 &#176;C and allow it to solubilize for 1-2 h. Pipet the DNA up and down to speed up the hydration process. 
	&#8203;NOTE: Add more DNA hydration solution if the sample appears too viscous.</li>
</ol>
3. Cell-free DNA extraction
<ol>
	<li>The amount of supernatant from step 1.5 is variable. Add 70 &#181;L of conditioning buffer (see Table of Materials) for every milliliter of the supernatant.</li>
	<li>Mix clearing beads well by vortexing. Add 10 &#181;L of clearing beads if processing &#60;14 mL of cell-free supernatant, 20 &#181;L if processing 14-40 mL.</li>
	<li>Mix sample/bead mixture well by vortexing. Centrifuge at 3,000 x g for 15 min at room temperature.</li>
	<li>Without disturbing the pellet, pipette out the supernatant leaving behind 100 &#181;L if 10 &#181;L of clearing beads are used, and 200 &#181;L if 20 &#181;L of clearing beads are used.</li>
	<li>Add an equal volume of Urine Pellet Digestion buffer (see Table of Materials) to the pellet and resuspend the pellet well by vortexing or pipetting.</li>
	<li>Add Proteinase K (5% (v/v)) to the resuspended pellet (e.g., add 10 &#181;L of Proteinase K to 200 &#181;L mixture) and mix well by gentle vortexing.</li>
	<li>Incubate the pellet mixture at 55 &#176;C for 30 min.</li>
	<li>Add one volume of Genomic lysis buffer (see Table of Materials) to the digestion mixture (e.g., 210 &#181;L Genomic lysis buffer to 210 &#181;L of digestion mixture) and mix by vortexing.</li>
	<li>Transfer the commercially available spin column (see Table of Materials) to a new collection tube.</li>
	<li>Add 200 &#181;L of Urine DNA Prep buffer to the spin column, centrifuge at &#8805;16,000 x g for 1 min at room temperature, and discard flow-through.</li>
	<li>Add 700 &#181;L of Urine DNA wash buffer to the column, centrifuge at &#8805;16,000 x g for 1 min at room temperature and discard flow-through.</li>
	<li>Repeat the step with 200 &#181;L Urine DNA wash buffer.</li>
	<li>Transfer the spin column to a DNase/RNase-free microcentrifuge tube.</li>
	<li>Add the appropriate volume of DNA elution buffer (based on the molecular tests to be performed) directly onto the column matrix and allow it to stand for 3-5 min at room temperature. 
	NOTE: 30 &#181;L of elution buffer was used in the example cases. More elution buffer is used if additional testing is required.</li>
	<li>Centrifuge at &#8805;16,000 x g at room temperature for 1 min to obtain the cell-free DNA.</li>
</ol>

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

<ol>
	<li>Chan, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+vitreoretinal+lymphoma:+a+report+from+an+International+Primary+Central+Nervous+System+Lymphoma+Collaborative+Group+symposium.">Primary vitreoretinal lymphoma: a report from an International Primary Central Nervous System Lymphoma Collaborative Group symposium.</a> Oncologist. 16 (11), 1589-1599 (2011).</li><li>Sagoo, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+intraocular+lymphomas.">Primary intraocular lymphomas.</a> Clinical &amp; Experimental Ophthalmology. 59 (5), 503-516 (2014).</li><li>Coupland, S. E., Damato, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+intraocular+lymphomas.">Understanding intraocular lymphomas.</a> Clinical &amp; Experimental Ophthalmology. 36 (6), 564-578 (2008).</li><li>Cassoux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+and+central+nervous+system+lymphoma:+clinical+features+and+diagnosis.">Ocular and central nervous system lymphoma: clinical features and diagnosis.</a> Ocular Immunology and Inflammation. 8 (4), 243-250 (2000).</li><li>Read, R. W., Zamir, E., Rao, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+masquerade+syndrome.">Neoplastic masquerade syndrome.</a> Survey Ophthalmology. 47, 81-124 (2002).</li><li>Carbonell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+recommendations+for+the+diagnosis+of+vitreoretinal+lymphoma.">Consensus recommendations for the diagnosis of vitreoretinal lymphoma.</a> Ocular Immunology and Inflammation. 23 (3), 507-520 (2021).</li><li>Demirci, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+humor-derived+MYD88+L265P+mutation+analysis+in+vitreoretinal+lymphoma:+a+potential+less+invasive+method+for+diagnosis+and+treatment+response+assessment.">Aqueous humor-derived MYD88 L265P mutation analysis in vitreoretinal lymphoma: a potential less invasive method for diagnosis and treatment response assessment.</a> Ophthalmology Retina. 7 (2), 189-195 (2023).</li><li>Machemer, R., Buettner, H., Norton, E. W., Parel, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrectomy:+a+pars+plana+approach.">Vitrectomy: a pars plana approach.</a> Transactions - American Academy of Ophthalmology and Otolaryngology. 75 (4), 813-820 (1971).</li><li>Vogel, M. H., Font, R. L., Zimmerman, L. E., Levine, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reticulum+cell+sarcoma+of+the+retina+and+uvea.+Report+of+sex+cases+and+review+of+the+literature.">Reticulum cell sarcoma of the retina and uvea. Report of sex cases and review of the literature.</a> American Journal of Ophthalmology. 66 (2), 205-215 (1968).</li><li>Kimura, K., Usui, Y., Goto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Japanese+intraocular+lymphoma+study+group.+clinical+features+and+diagnostic+significance+of+the+intraocular+fluid+of+217+patients+with+intraocular+lymphoma.">Japanese intraocular lymphoma study group. clinical features and diagnostic significance of the intraocular fluid of 217 patients with intraocular lymphoma.</a> Japanese Journal of Ophthalmology. 56 (4), 383-389 (2012).</li><li>Davis, J. L., Miller, D. M., Ruiz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+testing+of+vitrectomy+specimens.">Diagnostic testing of vitrectomy specimens.</a> American Journal of Ophthalmology. 140 (5), 822-829 (2005).</li><li>Char, D. H., Ljung, B. M., Miller, T., Phillips, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+intraocular+lymphoma+(ocular+reticulum+cell+sarcoma)+diagnosis+and+management.">Primary intraocular lymphoma (ocular reticulum cell sarcoma) diagnosis and management.</a> Ophthalmology. 95 (5), 625-630 (1988).</li><li>Tanaka, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+accurate+diagnosis+of+vitreoretinal+lymphoma+using+a+combination+of+diagnostic+test+results:+a+prospective+observational+study.">More accurate diagnosis of vitreoretinal lymphoma using a combination of diagnostic test results: a prospective observational study.</a> Ocular Immunology and Inflammation. 30 (6), 1354-1360 (2022).</li><li>Missotten, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+immunophenotyping+is+a+valuable+tool+for+detection+of+intraocular+lymphoma.">Multicolor flow cytometric immunophenotyping is a valuable tool for detection of intraocular lymphoma.</a> Ophthalmology. 120 (5), 991-996 (2013).</li><li>Bertram, H. C., Check, I. J., Milano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping+large+B-cell+lymphomas.+Flow+cytometric+pitfalls+and+pathologic+correlation.">Immunophenotyping large B-cell lymphomas. Flow cytometric pitfalls and pathologic correlation.</a> American Journal of Clinical Pathology. 116 (2), 191-203 (2001).</li><li>Bonzheim, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+hallmarks+of+primary+and+secondary+vitreoretinal+lymphoma.">The molecular hallmarks of primary and secondary vitreoretinal lymphoma.</a> Blood Advances. 6 (5), 1598-1607 (2022).</li><li>Shi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+of+the+high+prevalence+of+MYD88+L265P+mutated+vitreoretinal+lymphoma+identified+by+droplet+digital+polymerase+chain+reaction.">Clinical relevance of the high prevalence of MYD88 L265P mutated vitreoretinal lymphoma identified by droplet digital polymerase chain reaction.</a> Ocular Immunology and Inflammation. 29 (3), 448-455 (2021).</li><li>Bustamante, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+tracking+through+driver+mutations+as+a+liquid+biopsy-based+marker+for+uveal+melanoma.">Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based marker for uveal melanoma.</a> Journal of Experimental and Clinical Cancer Research. 40 (1), 196 (2021).</li></ol>

The authors have nothing to disclose.

Vitreoretinal lymphoma (VRL) is an aggressive large B-cell lymphoma1,2,3 whose symptoms can mimic other vitreoretinal diseases4,5. Molecular testing of vitreous and, more recently, aqueous humor has become a critical method for making the diagnosis of VRL or ruling it out. However, these fluids are very low in volume and often have low cellularity. Many of the cells with...

Vitreoretinal lymphoma (VRL) is an aggressive lymphoma associated with primary central nervous system diffuse large B-cell lymphoma1,2,3. VRL is typically fatal due to its involvement in the central nervous system1,2. Although rare1,4, VRL often presents with symptoms similar to posterior uveitis and other vitreoretinal diseases4,5. Consequently, patients exhibiting uveitis symptoms require a diagnosis to either confirm or rule out VRL.
Recently, consensus criteria for diagnosing VRL were published, which involve a combination of clinical examination and laboratory findings6. Specimens commonly used to diagnose VRL include vitreous humor and, more recently, aqueous humor7. Vitreous humor is obtained through a surgical procedure called pars plana vitrectomy, which allows access to the posterior segment of the eye8.
In the presented protocol, both aqueous humor and vitreous humor specimens were collected for cellular and cfDNA extraction. After anesthetizing the patients and placing trocars approximately 4 mm from the corneal limbus, an aqueous humor sample of approximately 100-200 &#181;L was obtained using a 1 mL tuberculin syringe at the corneal limbus. For pseudophakic patients, undiluted vitreous was obtained by introducing sterile air into the infusion, enabling the collection of a larger amount of undiluted vitreous (up to 3.5 mL). In phakic patients, approximately 500 to 1000 &#181;L of undiluted vitreous was removed before turning on an infusion of balanced salt solution. In some cases, secondarily diluted vitreous (500 to 2,000 &#181;L) was collected by switching the infusion to fluid and placing the vitrector within the vitreous skirt to obtain this sample. The most dilute vitreous fraction was collected by preserving the cassette bag (Supplemental Figure 1) at the end of the surgery. Once this bag reached the pathology department, dilute vitreous was obtained from draining fluid out of this bag into conical tubes for subsequent DNA extraction.
Cytopathology of vitreous fluid is often considered the gold standard9. However, several studies have demonstrated limited sensitivity due to processing and minimal cellularity10,11,12. Flow cytometry can aid in identifying clonal B-cells but can also be limited by low cellularity and the fragility of large lymphoma cells13,14,15. Both cytopathology and flow cytometry requires intact whole cells. Many of these cells are destroyed during collection, storage, and processing. When molecular testing is performed using DNA extracted from intact cells (cellular DNA), it suffers from this same limitation. In addition, dividing the limited vitreous specimen for all of these tests reduces the amount of material available for each test.
Cell-free DNA (cfDNA) represents another source of DNA that does not require intact cells. cfDNA from vitreous specimens has been used for the detection of VRL16,17 as well as uveal melanoma18. In this protocol, cellular and cell-free DNA are extracted from vitreous and aqueous fluid to detect VRL.

<table><tbody><tr><td>2-Propanol (Isopropanol)</td><td>Fischer</td><td>A415-500</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator-10</td><td>Zymo Research</td><td>D4011</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator-5</td><td>Zymo Research</td><td>D4003</td><td></td></tr><tr><td>Gentra Puregene Cell Lysis Solution</td><td>Qiagen</td><td>158906</td><td></td></tr><tr><td>Gentra Puregene DNA Hydration Solution</td><td>Qiagen</td><td>158916</td><td></td></tr><tr><td>Gentra Puregene Protein Precipitation Solution</td><td>Qiagen</td><td>158912</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P-4417</td><td></td></tr><tr><td>Quick-DNA Urine Kit</td><td>Zymo Research</td><td>D3061</td><td>Conditioning buffer; also includes clearing beads, Proteinase K and spin columns</td></tr><tr><td>Ultrapure Glycogen, 20 &amp;micro;g/&amp;micro;L (20 mg/mL)</td><td>Thermo Fisher/Invitrogen</td><td>10814010</td><td></td></tr></tbody></table>

cell-free dna extraction of vitreous and aqueous humor specimens for diagnosis and monitoring of vitreoretinal lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

These extraction methods were performed on a limited number of cases to ensure adequate yield and amplifiability of cell-free DNA in comparison with cellular DNA from the vitreous (four) and aqueous (four) specimens. From these samples, DNA yields from the cell-free component of these fluids are similar to that of the cellular component (Table 1). Cellular and cfDNA from these samples were also evaluated using molecular testing, such as for the VRL-associated mutation MYD88 L265P. An allele-spec...

Watch this Scientific Journal Video about Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor at JoVE.com

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...

cell-free-dna-extraction-vitreous-aqueous-humor-specimens-for

Research

JoVE Journal

Cancer Research

1.9K Views.  University of Michigan, Ann Arbor. Met dit onderzoek proberen we moleculaire tests van glasvocht uit het oog te gebruiken door vitrectomie om vitreoretinaal lymfoom of VRL te diagnosticeren, een agressieve ziekte waarvan de symptomen overlappen met die van veel voorkomende inflammatoire oogaandoeningen. Cytopathologie, flowcytometrie en moleculair testen zijn huidige methoden die worden gebruikt om VRL te diagnosticeren. Gepubliceerde gevoeligheden van niet-moleculaire methoden zijn 44 tot 90% voor cytopathologie en 50 tot 60%...

Video: Auteur in de schijnwerpers: VRL-diagnose bevorderen met behulp van celvrije DNA-extractie uit glasvocht 

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Medicine

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Bioengineering

Aqueous Humor as a Liquid Biopsy for Retinoblastoma: Clear Corneal Paracentesis and Genomic Analysis

There is significant potential clinical utility for the application of a liquid biopsy platform for retinoblastoma, given that direct tumor biopsy is prohibited in these patients. The aqueous humor (AH) forms in a separate compartment from the tumor but is enclosed within the same ocular space. Thus, it is an enriched source of eye-specific tumoral genomic information that can be used as a liquid biopsy or surrogate to tumor biopsy for this disease. This manuscript details a methodology for safely extracting the AH from retinoblastoma eyes via clear corneal paracentesis. Additionally, the steps for genomic analysis, including cell-free DNA isolation and purification, next-generation sequencing, somatic copy number alteration (SCNA) analysis, RB1 single nucleotide variant (SNV) mutation identification, and tumor fraction estimation are presented. The pre-analytical, analytical, and early clinical validity of the AH liquid biopsy platform have been evaluated; however, it is not without limitations. These are largely a consequence of the quantity of cell-free DNA that is required for certain steps of the assay. Compared to other blood-based liquid biopsy platforms currently under investigation for retinoblastoma, an AH-based platform is limited by the volume of biofluid (and thus the quantity of DNA) that can be extracted from the eye; the benefit is that AH is eye-specific. The platform discussed here is unique in that it detects circulating tumor DNA in the AH via two mechanisms (SCNAs and RB1 SNVs), yielding a higher sensitivity for identifying tumoral genomic information. The AH liquid biopsy has the potential for direct clinical application to precision oncology for retinoblastoma patients, with particular importance for patients with bilateral disease as the AH is specific to the tumors in each eye. There is ongoing research with applications of this platform to patients with other ocular tumors as well.

The aqueous humor is a high-yield liquid biopsy for retinoblastoma, intraocular cancer that cannot be biopsied in vivo due to the risk of extraocular spread. Herein, a method for safely extracting aqueous humor via clear corneal paracentesis and steps for genomic analysis to identify prognostic biomarkers are presented.

There is significant potential clinical utility for the application of a liquid biopsy platform for retinoblastoma, given that direct tumor biopsy is ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Immunology and Infection

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the research laboratory. Here, we developed a protocol for acquiring high-quality liquid biopsies for molecular analyses from the aqueous humor and the vitreous from patients undergoing eye surgery. In this workflow, a Mobile Operating Room Lab Interface (MORLI) cart equipped with a computer, a barcode scanner, and lab instruments, including onboard cold storage, is used to obtain and archive human biological samples. A web-based data privacy-compliant database enables annotating each sample over its lifetime, and a cartesian coordinate system allows tracking each barcoded specimen in storage, enabling quick and accurate retrieval of samples for downstream analyses. Molecular characterization of human tissue samples not only serves as a diagnostic tool (e.g., to distinguish between infectious endophthalmitis and other non-infectious intraocular inflammation) but also represents an important component of translational research, allowing the identification of new drug targets, development of new diagnostic tools, and personalized therapeutics.

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

A critical challenge in translational research is establishing a viable and efficient interface between patient care in the operating room (OR) and the ...

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

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